Multiplexed Functional Assessments of MYH7 Variants in Human Cardiomyocytes.

BACKGROUND: Pathogenic autosomal-dominant missense variants in MYH7 (myosin heavy chain 7), which encodes the sarcomeric protein (ß-MHC [beta myosin heavy chain]) expressed in cardiac and skeletal myocytes, are a leading cause of hypertrophic cardiomyopathy and are clinically actionable. However, ≈75% of MYH7 missense variants are of unknown significance. While human-induced pluripotent stem cells (hiPSCs) can be differentiated into cardiomyocytes to enable the interrogation of MYH7 variant effect in a disease-relevant context, deep mutational scanning has not been executed using diploid hiPSC derivates due to low hiPSC gene-editing efficiency. Moreover, multiplexable phenotypes enabling deep mutational scanning of MYH7 variant hiPSC-derived cardiomyocytes are unknown. METHODS: To overcome these obstacles, we used CRISPRa On-Target Editing Retrieval enrichment to generate an hiPSC library containing 113 MYH7 codon variants suitable for deep mutational scanning. We first established that ß-MHC protein loss occurs in a hypertrophic cardiomyopathy human heart with a pathogenic MYH7 variant. We then differentiated the MYH7 missense variant hiPSC library to cardiomyocytes for multiplexed assessment of ß-MHC variant abundance by massively parallel sequencing and hiPSC-derived cardiomyocyte survival. RESULTS: Both the multiplexed assessment of ß-MHC abundance and hiPSC-derived cardiomyocyte survival accurately segregated all known pathogenic variants from synonymous variants. Functional data were generated for 4 variants of unknown significance and 58 additional MYH7 missense variants not yet detected in patients. CONCLUSIONS: This study leveraged hiPSC differentiation into disease-relevant cardiomyocytes to enable multiplexed assessments of MYH7 missense variants for the first time. Phenotyping strategies used here enable the application of deep mutational scanning to clinically actionable genes, which should reduce the burden of variants of unknown significance on patients and clinicians.

Cardiomyocyte nuclear remodeling after mechanical unloading.

Cardiomyocytes increase DNA content in response to stress in humans. DNA content is reported to decrease in association with increased markers of proliferation in cardiomyocytes following left ventricular assist device (LVAD) unloading. However, cardiac recovery resulting in LVAD explant is rare. Thus, we sought to test the hypothesis that changes in DNA content with mechanical unloading occurs independent of cardiomyocyte proliferation by quantifying cardiomyocyte nuclear number, cell size, DNA content, and the frequency of cell-cycling markers using a novel imaging flow cytometry methodology comparing human subjects undergoing LVAD implantation or primary transplantation. We found that cardiomyocyte size was 15% smaller in unloaded versus loaded samples without differences in the percentage of mono-, bi-, or multinuclear cells. DNA content per nucleus was significantly decreased in unloaded hearts versus loaded controls. Cell-cycle markers, Ki67 and phospho-histon3 (H3P), were not increased in unloaded samples. In conclusion, unloading of failing hearts is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell-cycle markers, they may represent a regression of hypertrophic nuclear remodeling and not proliferation.NEW & NOTEWORTHY Our data suggest that increases in DNA content that occur with cardiomyocyte hypertrophy in heart failure may reverse with mechanical unloading.

Tropoelastin Improves Post-Infarct Cardiac Function.

BACKGROUND: Myocardial infarction (MI) is among the leading causes of death worldwide. Following MI, necrotic cardiomyocytes are replaced by a stiff collagen-rich scar. Compared to collagen, the extracellular matrix protein elastin has high elasticity and may have more favorable properties within the cardiac scar. We sought to improve post-MI healing by introducing tropoelastin, the soluble subunit of elastin, to alter scar mechanics early after MI. METHODS AND RESULTS: We developed an ultrasound-guided direct intramyocardial injection method to administer tropoelastin directly into the left ventricular anterior wall of rats subjected to induced MI. Experimental groups included shams and infarcted rats injected with either PBS vehicle control or tropoelastin. Compared to vehicle treated controls, echocardiography assessments showed tropoelastin significantly improved left ventricular ejection fraction (64.7±4.4% versus 46.0±3.1% control) and reduced left ventricular dyssynchrony (11.4±3.5 ms versus 31.1±5.8 ms control) 28 days post-MI. Additionally, tropoelastin reduced post-MI scar size (8.9±1.5% versus 20.9±2.7% control) and increased scar elastin (22±5.8% versus 6.2±1.5% control) as determined by histological assessments. RNA sequencing (RNAseq) analyses of rat infarcts showed that tropoelastin injection increased genes associated with elastic fiber formation 7 days post-MI and reduced genes associated with immune response 11 days post-MI. To show translational relevance, we performed immunohistochemical analyses on human ischemic heart disease cardiac samples and showed an increase in tropoelastin within fibrotic areas. Using RNA-seq we also demonstrated the tropoelastin gene ELN is upregulated in human ischemic heart disease and during human cardiac fibroblast-myofibroblast differentiation. Furthermore, we showed by immunocytochemistry that human cardiac fibroblast synthesize increased elastin in direct response to tropoelastin treatment. CONCLUSIONS: We demonstrate for the first time that purified human tropoelastin can significantly repair the infarcted heart in a rodent model of MI and that human cardiac fibroblast synthesize elastin. Since human cardiac fibroblasts are primarily responsible for post-MI scar synthesis, our findings suggest exciting future clinical translation options designed to therapeutically manipulate this synthesis.

Generation of human iPSC line from an arrhythmogenic cardiomyopathy patient with a DSP protein-truncating variant.

Arrhythmogenic cardiomyopathy is an inheritable heart disease characterized by lethal heart rhythms and abnormal contractile function. Mutations in desmoplakin (DSP), a protein linking the cardiac desmosome with intermediate filaments, are associated with arrhythmogenic cardiomyopathy. Here we generated a human induced pluripotent stem cell (hiPSC) line from a patient with a heterozygous protein-truncating variant in DSP (c.1386del Leu462Serfs*22). This line has a normal karyotype and expression of pluripotency markers, and can differentiate into all three germ layers. This line is well suited for in vitro mechanistic studies of mechanism of DSP protein-truncation mutations in the context of arrhythmogenic cardiomyopathy.

MBNL1 drives dynamic transitions between fibroblasts and myofibroblasts in cardiac wound healing.

Dynamic fibroblast to myofibroblast state transitions underlie the heart's fibrotic response. Because transcriptome maturation by muscleblind-like 1 (MBNL1) promotes differentiated cell states, this study investigated whether tactical control of MBNL1 activity could alter myofibroblast activity and fibrotic outcomes. In healthy mice, cardiac fibroblast-specific overexpression of MBNL1 transitioned the fibroblast transcriptome to that of a myofibroblast and after injury promoted myocyte remodeling and scar maturation. Both fibroblast- and myofibroblast-specific loss of MBNL1 limited scar production and stabilization, which was ascribed to negligible myofibroblast activity. The combination of MBNL1 deletion and injury caused quiescent fibroblasts to expand and adopt features of cardiac mesenchymal stem cells, whereas transgenic MBNL1 expression blocked fibroblast proliferation and drove the population into a mature myofibroblast state. These data suggest MBNL1 is a post-transcriptional switch, controlling fibroblast state plasticity during cardiac wound healing.

Long-term adult congenital heart disease survival after heart transplantation: A restricted mean survival time analysis.

BACKGROUND: Adult Congenital Heart Disease (ACHD) heart transplant recipients may have lower post-transplant survival resulting from higher peri-operative mortality than non-ACHD patients. However, the late risk of mortality appears lower in ACHD recipients. This study seeks to establish whether long-term heart transplant survival is reduced among ACHD recipients relative to non-ACHD recipients. METHODS: Adult patients who received a heart transplant between January, 2000 and December, 2019 in the United Network for Organ Sharing database were stratified by the presence of ACHD. Propensity-matched cohorts (1:4) were created to adjust for differences between groups. Graft survival at time points from 1 to 18 years was compared between groups using restricted mean survival time (RMST) analysis. RESULTS: The matched cohort included 1,139 ACHD and 4,293 non-ACHD patients. Median age was 35 years and 61% were male. Average survival time at 1 year was 0.85 years for ACHD patients and 0.93 years for non-ACHD patients (average difference: -0.08 years, 95% Confidence Interval [CI] -0.10 to -0.06, p < 0.001), reflecting higher immediate post-transplant mortality. Average survival time at 18 years was not clinically or statistically different: 11.14 years for ACHD patients and 11.40 years for non-ACHD patients (average difference: -0.26 years, 95% CI: -0.85 to + 0.32 years, p = 0.38). CONCLUSIONS: Despite increased medium-term mortality among ACHD patients after heart transplant, differences in long-term survival are minimal. Allocation of hearts to ACHD patients results in acceptable utility of donor hearts.

Boosting NAD level suppresses inflammatory activation of PBMCs in heart failure.

BACKGROUNDWhile mitochondria play an important role in innate immunity, the relationship between mitochondrial dysfunction and inflammation in heart failure (HF) is poorly understood. In this study we aimed to investigate the mechanistic link between mitochondrial dysfunction and inflammatory activation in peripheral blood mononuclear cells (PBMCs), and the potential antiinflammatory effect of boosting the NAD level.METHODSWe compared the PBMC mitochondrial respiration of 19 hospitalized patients with stage D HF with that of 19 healthy participants. We then created an in vitro model of sterile inflammation by treating healthy PBMCs with mitochondrial damage-associated molecular patterns (MitoDAMPs) isolated from human heart tissue. Last, we enrolled patients with stage D HF and sampled their blood before and after taking 5 to 9 days of oral nicotinamide riboside (NR), a NAD precursor.RESULTSWe demonstrated that HF is associated with both reduced respiratory capacity and elevated proinflammatory cytokine gene expressions. In our in vitro model, MitoDAMP-treated PBMCs secreted IL-6 that impaired mitochondrial respiration by reducing complex I activity. Last, oral NR administration enhanced PBMC respiration and reduced proinflammatory cytokine gene expression in 4 subjects with HF.CONCLUSIONThese findings suggest that systemic inflammation in patients with HF is causally linked to mitochondrial function of the PBMCs. Increasing NAD levels may have the potential to improve mitochondrial respiration and attenuate proinflammatory activation of PBMCs in HF.TRIAL REGISTRATIONClinicalTrials.gov NCT03727646.FUNDINGThis study was funded by the NIH, the University of Washington, and the American Heart Association.

Comparison of Neurologic Event Rates Among HeartMate II, HeartMate 3, and HVAD.

Strokes remain a leading cause of morbidity and mortality in patients with ventricular assist devices (VADs). Varying study populations, event definitions, and reporting methods make direct comparison of neurologic event risk across clinical trials and registries challenging. We aim to highlight important differences among major VAD studies and standardize rates of neurologic events to facilitate a comprehensive and objective comparison. We systematically identified and analyzed key clinical trials and registries evaluating the HeartMate II (HMII), HeartMate 3 (HM3), and HVAD devices. Reported neurologic events were nonexclusively categorized into ischemic stroke, hemorrhagic stroke, disabling stroke, fatal stroke, and other neurologic events per the studies' definitions. Event rates were standardized to events per patient-year (EPPY) and freedom from event formats. Seven key clinical trials and registries were included in our analysis. There is significant variation and overlap in neurologic event rates for the three VAD platforms across clinical trials (all neurologic events [EPPY]: HM3 0.17-0.21; HMII 0.19-0.26; HVAD 0.16-0.28). None performs consistently better for all types of neurologic events. Furthermore, stroke rates among VAD trials correlated with baseline stroke risk factors including ischemic etiology, history of atrial fibrillation, and history of prior stroke.

Accuracy of Doppler blood pressure measurement in continuous-flow left ventricular assist device patients.

AIMS: Accurate blood pressure (BP) measurement in continuous-flow ventricular assist device (CF-VAD) patients is imperative to reduce stroke risk. This study assesses the accuracy of the Doppler opening pressure method compared with the gold standard arterial line method in CF-VAD patients. METHODS AND RESULTS: In a longitudinal cohort of HeartMate II and HVAD patients, arterial line BP and simultaneously measured Doppler opening pressure were obtained. Overall correlation, agreement between Doppler opening pressure and arterial line mean vs. systolic pressure, and the effect of arterial pulsatility on the accuracy of Doppler opening pressure were analysed. A total of 1933 pairs of Doppler opening pressure and arterial line pressure readings within 1 min of each other were identified in 154 patients (20% women, mean age 55 ± 15, 50% HeartMate II and 50% HVAD). Doppler opening pressure had good correlation with invasive mean arterial pressure (r = 0.742, P < 0.0001) and more closely approximated mean than systolic BP (mean error 2.4 vs. -8.4 mmHg). Arterial pulsatility did not have a clinically significant effect on the accuracy of the Doppler opening pressure method. CONCLUSIONS: Doppler opening pressure should be the standard non-invasive method of BP measurement in CF-VAD patients.

Long-Term Right Ventricular Assist Device Therapy in an Adult with Pulmonary Atresia/Intact Ventricular Septum.

Durable ventricular assist device (VAD) support is uncommonly employed in adult congenital heart disease and often involves supporting a systemic right ventricle (RV). Ventricular assist device support of a subpulmonic RV is even more unusual.

Durable mechanical circulatory support in teenagers and adults with congenital heart disease: A systematic review.

BACKGROUND: Heart failure is the leading cause of morbidity and mortality for adults with congenital heart disease (ACHD). Many patients are ineligible for transplantation, and those who are eligible often face long wait times with high wait-list morbidity. Durable mechanical circulatory support (MCS) may be an option for many patients. This systematic review evaluates the published literature on the use of durable MCS in teenagers and adults with congenital heart disease. METHODS: A comprehensive search of MEDLINE (PubMed), EMBASE, and the Cochrane Library was performed electronically in July 2015 and updated in March 2016, guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. RESULTS: Individual case reports and several case series identified 66 patients with ACHD treated with durable MCS. More than half were INTERMACS 1 or 2 at the time of implantation. Patients with Fontan repairs were more frequently classified as INTERMACS 1 or 2 (89% compared to 59% or less among other groups). Cases published after 2010 showed a trend toward less severe INTERMACS status, and patients were less likely to have received transplants by the time of reporting (31% compared to 61% prior). Durable MCS was implanted as bridge-to-transplant in 77%. Patients with Fontan repair accounted for 14% of cases. CONCLUSION: Reports of durable MCS utilization in patients with ACHD are becoming more frequent and devices are being implanted in more stable patients. Reports are mostly case reports or small case series so reporting bias is likely and prospective protocoled reporting is needed.

Cell-Specific Pathways Supporting Persistent Fibrosis in Heart Failure.

BACKGROUND: Only limited data exist describing the histologic and noncardiomyocyte function of human myocardium in end-stage heart failure (HF). OBJECTIVES: The authors sought to determine changes in noncardiomyocyte cellular activity in patients with end-stage HF after left ventricular assist device (LVAD)-induced remodeling to identify mechanisms impeding recovery. METHODS: Myocardium was obtained from subjects undergoing LVAD placement and/or heart transplantation. Detailed histological analyses were performed, and, when feasible, mononuclear cells were isolated from fresh, dissociated myocardium for quantitative reverse transcription polymerase chain reaction studies. Echocardiographic and catheterization data were obtained during routine care. RESULTS: Sixty-six subjects were enrolled; 54 underwent 8.0 ± 1.2 months of LVAD unloading. Despite effective hemodynamic unloading and remodeling, there were no differences after LVAD use in capillary density (0.78 ± 0.1% vs. 0.9 ± 0.1% capillary area; n = 42 and 28, respectively; p = 0.40), cardiac fibrosis (25.7 ± 2.4% vs. 27.9 ± 2.4% fibrosis area; n = 44 and 31, respectively; p = 0.50), or macrophage density (80.7 ± 10.4 macrophages/mm2 vs. 108.6 ± 15 macrophages/mm2; n = 33 and 28, respectively; p = 0.1). Despite no change in fibrosis or myofibroblast density (p = 0.40), there was a 16.7-fold decrease (p < 0.01) in fibroblast-specific collagen expression. Furthermore, there was a shift away from pro-fibrotic/alternative pro-fibrotic macrophage signaling after LVAD use. CONCLUSIONS: Despite robust cardiac unloading, capillary density and fibrosis are unchanged compared with loaded hearts. Fibroblast-specific collagen expression was decreased and might be due to decreased stretch and/or altered macrophage polarization. Dysfunctional myocardium may persist, in part, from ongoing inflammation and poor extracellular matrix remodeling. Understanding these changes could lead to improved therapies for HF.

Heart failure with preserved ejection fraction and skeletal muscle physiology.

Heart failure with preserved ejection fraction (HFpEF) accounts for half of all heart failure in the USA, increases in prevalence with aging, and has no effective therapies. Intriguingly, the pathophysiology of HFpEF has many commonalities with the aged cardiovascular system including reductions in diastolic compliance, chronotropic defects, increased resistance in the peripheral vasculature, and poor energy substrate utilization. Decreased exercise capacity is a cardinal symptom of HFpEF. However, its severity is often out of proportion to changes in cardiac output. This observation has led to studies of muscle function in HFpEF revealing structural, biomechanical, and metabolic changes. These data, while incomplete, support a hypothesis that similar to aging, HFPEF is a systemic process. Understanding the mechanisms leading to exercise intolerance in this condition may lead to strategies to improve morbidity in both HFpEF and aging.

Cardiac macrophages adopt profibrotic/M2 phenotype in infarcted hearts: Role of urokinase plasminogen activator.

BACKGROUND: Macrophages (mac) that over-express urokinase plasminogen activator (uPA) adopt a profibrotic M2 phenotype in the heart in association with cardiac fibrosis. We tested the hypothesis that cardiac macs are M2 polarized in infarcted mouse and human hearts and that polarization is dependent on mac-derived uPA. METHODS: Studies were performed using uninjured (UI) or infarcted (MI) hearts of uPA overexpressing (SR-uPA), uPA null, or nontransgenic littermate (Ntg) mice. At 7days post-infarction, cardiac mac were isolated, RNA extracted and M2 markers Arg1, YM1, and Fizz1 measured with qrtPCR. Histologic analysis for cardiac fibrosis, mac and myofibroblasts was performed at the same time-point. Cardiac macs were also isolated from Ntg hearts and RNA collected after primary isolation or culture with vehicle, IL-4 or plasmin and M2 marker expression measured. Cardiac tissue and blood was collected from humans with ischemic heart disease. Expression of M2 marker CD206 and M1 marker TNFalpha was measured. RESULTS: Macs from WT mice had increased expression of Arg1 and Ym1 following MI (41.3±6.5 and 70.3±36, fold change vs UI, n=8, P<0.007). There was significant up-regulation of cardiac mac Arg1 and YM1 with MI in both WT and uPA null mice (n=4-9 per genotype and condition). Treatment with plasmin increased expression of Arg1 and YM1 in cultured cardiac macs. Histologic analysis revealed increased density of activated fibroblasts and M2 macs in SR-uPA hearts post-infarction with associated increases in fibrosis. Cardiac macs isolated from human hearts with ischemic heart disease expressed increased levels of the M2 marker CD206 in comparison to blood-derived macs (4.9±1.3). CONCLUSIONS: Cardiac macs in mouse and human hearts adopt a M2 phenotype in association with fibrosis. Plasmin can induce an M2 phenotype in cardiac macs. However, M2 activation can occur in the heart in vivo in the absence of uPA indicating that alternative pathways to activate plasmin are present in the heart. Excess uPA promotes increased fibroblast density potentially via potentiating fibroblast migration or proliferation. Altering macrophage phenotype in the heart is a potential target to modify cardiac fibrosis.